Collagen is a major component of the extracellular matrix (ECM). At least 27 genetically different collagen types have been identified, each containing at least one dominant collagenous domain (1). These collagenous domains have a characteristic triple helix structure formed by repeating Gly-X-Y sequences in each participating chain where X often is Proline and Y is hydroxyproline. The collagen monomers often assemble into more complex structures of varying organizations such as fibrils (types I-III, V and XI), networks (types IV, VIII and X) and beaded filaments (type VI) (2). The fibrillar collagen types I and III are the major structural components of the ECM of skin, cardiac and vascular tissues, whereas type II collagen is a major structural component of cartilage. In addition to contributing to the structural integrity of the tissues, collagens also affect cell behaviour through interactions with other matrix proteins and cellular receptors (3-6).
The integrins are a family of heterodimeric cell surface receptors involved in cell-cell and cell-substrate adhesion. They act as bridging molecules that link intracellular signaling molecules to the ECM through bi-directional signaling and control cell behaviour and tissue architecture (7). Four integrins, α1β1, α2β1, α10β1 and α11β1 have been shown to bind collagens (8-10). Of these, the α1β1 and α2β1 integrins have been studied In more detail compared to the others. Collagen integrin interactions play a role in normal and pathological physiology and directly affect cell adhesion, migration, proliferation and differentiation as well as angiogenesis, platelet aggregation and ECM assembly (11). However, the precise molecular mechanisms that lead to these activities are not understood.
Collagen binding by the four integrins is mediated by a ˜200 amino acids long so-called inserted domain (I domain) found between blades 2 and 3 of the β-propeller domain of the a chains. All four I domains (α1I, α2I, α10I, α11I) contain a metal ion-dependent adhesion site (MIDAS) that is required for coordinating a divalent cation and is essential for collagen binding. Synthetic collagen peptides containing the type I collagen derived sequences, GFOGER (SEQ ID NO: 1) or GLOGER (SEQ ID NO: 2) have been reported to bind with high affinity to α1I, α2I and α11I; furthermore, synthetic peptides containing these sequences inhibit the binding of I domains to intact collagens (12-14).
The crystal structures of apo-α2I and α2I in complex with a collagen peptide containing the GFOGER (SEQ ID NO: 1) sequence have been solved (15) and showed that the apo-α2I adopted an inactive “closed” conformation and the ligand bound α2I, an active “open” conformation (16). The Glu residue in the collagen peptide was shown in the structure of the complex to directly interact with a Mg2+ ion co-ordinated by the MIDAS motif and the Arg residue forms a salt bridge with D219 in α2I. The importance of the GER sequence in collagen for integrin binding was confirmed by mutagenesis studies, which showed that replacing Glu in the collagen peptide with an Asp residue completely abolished the binding whereas replacing the Arg with a Lys residue reduced the binding by 50% (17). The Phe residue in the collagen sequence appeared to participate in hydrophobic interactions with α2I and could be replaced by Leu. Both GFOGER (SEQ ID NO: 1) and GLOGER (SEQ ID NO: 2) bind to α1I and α2I (18). However, changing the Phe residue to a Met or an Ala reduced the apparent affinity of I domains (14). GASGER (SEQ ID NO: 3) was also reported to be recognized by the I domains but bound with lower affinity than GFOGER (SEQ ID NO: 1) and GLOGER (SEQ ID NO: 2) (13.14.18). Therefore, GFOGER (SEQ ID NO: 1) and GLOGER (SEQ ID NO: 2) are the only two known collagen-derived sequence motifs that support high affinity binding by the collagen-binding I domains. However, the GFOGER (SEQ ID NO: 1) and GLOGER (SEQ ID NO: 2) motifs are absent in some collagens such as human type III collagen. Additionally, previous studies have shown that CHO cell expressing α1β1 and α2β1 could adhere and spread on human type III collagen and furthermore, the recombinant proteins of α1I and α2I could bind to this collagen type (19).
Thus, there is a need in the art for understanding the mechanism by which type III collagen binds to integrins and in designing of integrin specific inhibitors. The present invention fulfills this long-standing need and desire in the art.